Melanoma is a progressive public health problem as its incidence continues to increase. Immunotherapy is now recognized to be an effective treatment for advanced melanoma, although it only benefits a small proportion of patients. The overall goal of this grant is to develop more effective strategies to use the immune system to recognize specific targets present on melanoma cells. We seek to build on prior accomplishments of this project in several specific ways to develop the most potent immunotherapeutic program. The first aim of the project is focused on investigating ways to stimulate a pathway known as GITR, present on T cells, as a way to stimulate effector T cells and inhibit suppressive T cells. In the second aim, different combinations of antibodies will be explored to find the optimal way to modulate different immunologic pathways. All of the immunomodulatory antibodies have clinical grade equivalents available or in development. Therefore, the results of this aim may have immediate clinical applicability. The best combination of antibodies will be combined with optimized DNA vaccine to assess if the chosen immune modulation program enhances the effect of the vaccine. The third aim will investigate the use of antigen specific T cells as a therapy for melanoma. It is known that infusions of both CD4+ (helper) and CD8+ (cytotoxic) T cells can have therapeutic benefit in melanoma patients. Determination of the optimal number and combination of such cells will be the goal of this part of the project. Once the details of the best combination of immunomodulating antibodies and adoptively transferred T cells are known, these will be combined in an attempt to develop a comprehensive immunotherapeutic program. This will be evaluated first in a transplanted tumor in mice. The fourth aim represents a continuing effort to generate a realistic model of spontaneous melanoma in mice. The same genetic aberrations present in human melanoma are being introduced into mice with melanocytes in the skin. This will represent the most stringent means to test the efficacy of the comprehensive immunotherapy program.